The machinery of DNA replication is remarkably conserved between species. Initiator protein recognizes the replication origin in a sequence-specific manner, and introduces DNA helicase onto DNA, which then continuously unwinds duplex DNA utilizing its ATP hydrolysis activity (Kornberg and Baker, 1992; Stillman, 1996). The loading of DNA helicase facilitates association of the DNA chain elongation machinery including DNA polymerases and primase. Single-stranded DNA binding (SSB) protein binds to the exposed DNA and facilitates the action of DNA polymerases. Topoisomerases help release the topological tension that accumulates on template DNA as a result of unwinding.

Figure 1. Assembly of replication forks at the replication origins in prokaryotes and eukaryotes. In prokaryotes (right), DnaA initiator protein first recognizes and binds to the origin (oriC) and this binding (in the presence of ATP) can induce local melting of the origin DNA within the AT-rich 13-mer sequences, to which DnaB–DnaC hetero-hexamers bind. After loading of DnaB protein, DnaC protein dissociates from the DNA, leaving DnaB protein at the forks. DnaB protein migrates in both directions on the template DNA, unwinding the duplex DNA. Primase interacts with translocating DnaB and lays down primer RNAs on lagging strand templates, which are elongated by the DNA polymerase III holoenzyme. In eukaryotes (left), ORC, in the presence of ATP, recognizes and binds to the origin, serving as a landing pad for pre-replicative components including MCM. Association of MCM with ORC is catalyzed by Cdt1 (not shown in this figure) and Cdc6. The major difference from prokaryotic origin activation is that the preRC needs to be activated by cell cycle signals in G1 for initial melting of the origin DNA to occur. This is achieved by phosphorylation events catalyzed by two distinct kinases, Cdc7 and CDK. This phosphorylation may trigger delivery of the helicase components at the forks, or simply activate the helicase activity of the preRC, establishing the active replication forks migrating in both directions. The phosphorylation events may also inactivate the replicative machinery to suppress refiring of the origins.

The eukaryotic proteins responsible for the above functions were first identified in studies on replication of DNA viruses (Challberg and Kelly, 1989). This lead to identification of proteins involved in the elongation stages of DNA replication, including DNA polymerases, polymerase-associated factors such as RFC, PCNA, and RFA (heterotrimeric SSB protein). Identification of those proteins critical for initiation depended upon the discovery of the origin recognition complex (ORC), which specifically recognized yeast chromosome replication origins in vitro (Bell and Stillman, 1992) (Fig. 1). Furthermore, genetic analyses in yeasts identified a number of mutant strains defective in maintenance of autonomously replicating sequences (ARS) plasmid or in the S phase. Mapping the mutations lead to identification of MCMs (Maine et al., 1984; Sinha et al., 1986) and Cdc45 (Moir et al., 1982; Hennessy et al., 1991; Hopwood and Dalton, 1996). Subsequent biochemistry with Xenopus egg extracts (Hua and Newport, 1998; Walter and Newport, 2000) demonstrated the critical roles of these new factors in the initiation process. We now know these proteins are widely conserved in eukaryotes.

In both prokaryotes and eukaryotes, initiation is the most critical regulatory step. In bacteria, DnaA binding is sufficient to cause duplex unwinding, triggering replication. In eukaryotes, formation of a prereplicative complex (preRC) at the origin cannot induce replication unless a further cue is provided from upstream cell cycle events. This cue is protein phosphorylation mediated by two essential serine-threonine kinases. One is the very well known cell cycle protein, cyclin-dependent kinase (CDK), and the other is the less popular Cdc7-Dbf4, which is gaining attention as an essential S phase regulator. In this article, we summarize progress in our understanding of the structure and function of this relatively new kinase member.

In the initial collections of Hartwell's cdc mutant strains, cdc7 was unique in that protein synthesis was no longer required for completion of the S phase after the functions of this protein were executed (Hartwell, 1971, 1973). dbf4 was isolated in a screen for mutant strains arresting with dumbbell shapes, indicative of a defect in initiation of DNA replication (Johnston and Thomas, 1982a,b). The terminal phenotypes of cdc7 and dbf4 are almost identical, suggesting that they may function in an identical or closely tied process. Cloning of the CDC7 gene indicated it encoded a serine-theronine kinase (Patterson et al., 1986). DBF4 was rediscovered as a multicopy suppressor of cdc7(ts), and it was shown that the Cdc7 kinase activity depended on Dbf4 (Yoon and Campbell, 1991; Kitada et al., 1992; Yoon et al., 1993). Furthermore, there is direct interaction between Cdc7 and Dbf4 (Dowell et al., 1994; Hardy and Pautz, 1996). Together, these studies strongly suggested that DBF4 encoded an activation subunit for the Cdc7 kinase. Recent reports indicate that Cdc7 is required for activation of each origin on the chromosomes throughout S phase (Bousset and Diffley, 1998; Donaldson et al., 1998). Thus, Cdc7 may be an ultimate triggerer of origin activation.

In spite of the essential roles of Cdc7-Dbf4 in initiation of DNA replication in budding yeast, proteins with homologous functions were not identified in other eukaryotic species, until hsk1+, encoding a fission yeast kinase with structural similarity to Cdc7, was identified (Masai et al., 1995). hsk1+ is essential for DNA replication in fission yeast and a null mutant displays premature mitosis in the absence of DNA replication. Hsk1 protein exhibits about 60% identity with budding yeast Cdc7 in the kinase conserved domains (Fig. 2), although its expression in the cdc7(ts) strain does not rescue temperature-sensitive growth. On the basis of structural similarity between Cdc7 and Hsk1, putative Cdc7 homologues from human, mouse, and Xenopus were isolated (Jiang and Hunter, 1997; Sato et al., 1999, 1997; Kim et al., 1998; Johnston et al., 1999; Masai et al., 2000; Masai and Arai, 2000b) (Table 1). The mammalian Cdc7 shares about 45% identity with yeast Cdc7 in the kinase domains (Fig. 2). The human homologue of the Cdc7 kinase (huCdc7) phosphorylated MCM subunits in vitro (Sato et al., 1997). However, these data alone did not prove this molecule represents the functional homologue of Cdc7.

Figure 2. Schematic drawing and comparison of Cdc7 and Dbf4-related subunits from humans, and both budding and fission yeasts. A: Comparison of Cdc7-related subunits. Yellow, red, and light blue segments represent, respectively, non-conserved N- and C-terminal region, conserved kinase domains, and less conserved kinase insert sequences. B: Comparison of Dbf4-related subunits. Yellow and red segments represent less conserved regions and conserved three Dbf4-motif sequences, respectively. In (B), “destruction box” indicates a “RXXL” motif, known to be targeted by the APC-dependent degradation pathway. Its functional significance has been experimentally shown in S. cerevisiae Dbf4, but not in ASK or Him1/Dfp1. Two putative nuclear localization signals (NLS) in ASK are also shown.

As in the case for budding yeast Cdc7, mammalian Cdc7 expressed singly in a recombinant form is inert as a kinase (Sato et al., 1997; Masai et al., 2000), although Hsk1 kinase has a substantial level of auto-phosphorylation activity on its own (Brown and Kelly, 1998; Takeda et al., 1999). Putative homologues for Dbf4 in fission yeast and mammals were identified through database or protein-interaction screening (Fig. 2, Table 1). A fission yeast homologue of Dbf4, dfp1+ was first reported by Brown and Kelly (1998) as an associating molecule in a purified Hsk1 kinase complex. Interestingly, Dfp1 does not significantly stimulate the autophosphorylation activity of Hsk1, but stimulates phosphorylation of exogenous substrates. This data led the authors to conclude that Dfp1 modifies the substrate specificity of Hsk1 kinase. The same molecule, named Him1, was identified independently through two-hybrid screening (Takeda et al., 1999). Dfp1/Him1 is essential for viability of fission yeast cells, and null mutant cells arrest with a 1C DNA content (Brown and Kelly, 1999; Takeda et al., 1999). The Dfp1/Him1 protein is mainly localized in triton-insoluble fractions, and can be dissolved by 0.15 M NaCl but not by nuclease treatment, indicating that it associates with some nuclear structures. A human Dbf4 homologue was identified by two-hybrid screening and was named ASK (activator for S phase kinase; Kumagai et al., 1999) or hsDbf4 (Jiang et al., 1999). Purification and characterization of the recombinant huCdc7-ASK complex demonstrated the role of ASK as an activation subunit for huCdc7 (Masai et al., 2000). Putative Dbf4 homologues have been reported also in Aspergillus, Drosophila, mouse, and Chinese hamster (James et al., 1999; Landis and Tower, 1999; Lepke et al., 1999; Guo and Lee, 2001).

Expression of budding yeast DBF4 is under cell cycle control and increases at late G1 through early S phases and decreases at the G2-M phase (Chapman and Johnston, 1989). The presence of a Mlu1 box in the promoter region of DBF4 suggests its transcription is regulated by the MBF transcription factor (Moll et al., 1992; Iyer et al., 2001). More recently, Dbf4 protein levels were shown to oscillate during the cell cycle. This oscillation appears to be mainly due to a change in the stability of the Dbf4 protein during the cell cycle. Dbf4 protein is stabilized in strains mutated in genes encoding APC subunits, indicating that APC-dependent degradation is responsible for the protein loss in the M-G1 phase (Oshiro et al., 1999; Weinreich and Stillman, 1999; Ferreira et al., 2000). Furthermore, mutations in the putative degradation box sequence present at the N-terminal encoding region of the gene can stabilize the Dbf4 protein (Ferreira et al., 2000) (Fig. 2).

Expression of fission yeast and mammalian Cdc7-regulatory subunits, Dfp1/Him1 and ASK, respectively, are also under similar cell cycle regulation (Brown and Kelly, 1999; Kumagai et al., 1999; Takeda et al., 1999). The protein level of Dfp1/Him1 is very low at the M-G1 phase and sharply increases at the G1/S boundary and stays at a high level during the S phase. Similarly, ASK protein level is very low at the M-G1 phase and increases at late G1, staying high during the S through G2 phase. Transcription of dfp1+/him1+ and ASK also oscillates during the cell cycle, increasing during G1 phase and decreasing by the G2 phase. The fact that proteins are not detected during the G1 phase, when transcription is already activated, indicates regulation at a protein level (Brown and Kelly, 1999; Takeda et al., 1999). The promoter region of dfp1+/him1+ does not contain a perfect match for the Mlu1 box sequence, and its transcript is detected in cdc10-arrested cells, indicating that dfp1+/him1+ transcription may not be under regulation of the Cdc10 transcription factor (Ogino et al., unpublished communications). The human ASK gene promoter region contains multiple E2F and Sp1 binding sites, and its transcription can be activated by ectopic expression of E2F factors. The promoter is repressed by growth factor or serum depletion, and growth stimulation activates transcription. This stimulation can be mediated by the promoter proximal 63 base pair containing a Sp1 site, but not canonical E2F binding sites (Yamada et al., unpublished communications).

Expression of catalytic subunits is relatively constant during the cell cycle and the protein appears to be stable (Sato et al., 1997; Takeda et al., 1999). The mRNA level during the proliferating cell cycle is fairly constant, although hsk1+ mRNA may undergo slight oscillation during the cell cycle (Takeda et al., 1999). Transcription of mammalian Cdc7 responds to growth stimulation as well as to E2F transcription factor (Kim et al., 1998), and the promoter proximal 231 base pair sequence containing three E2F sites and one Sp1 site is sufficient for this stimulation.

The kinase activity of Cdc7, which appears to be closely correlated with the level of the regulatory subunit, oscillates during the cell cycle (Kumagai et al., 1999). Activation of CDK is regulated via phosphorylation of the catalytic subunit on a conserved threonine residue in the T-loop by CAK (Draetta, 1997). Interestingly, substitution of the analogous Cdc7 residue with alanine results in significant reduction of the kinase activity and this residue can be phosphorylated by CDK in vitro (Buck et al., 1991; Ohtoshi et al., 1997; Masai et al., 2000). However, there is no direct evidence indicating phosphorylation of this Cdc7 threonine residue in vivo. Dfp1/Him1 as well as ASK proteins are hyper-phosphorylated during the S phase, presumably by autophosphorylation (Kumagai et al., 1999; Takeda et al., 1999, 2001). Hsk1 phosphorylates multiple serine/threonine residues on its associated regulatory subunits, although the functional significance of this is not clear.

Conservation between yeasts and higher eukaryotes of the primary structure of Cdc7 catalytic subunits strongly suggests the functional conservation of these kinases. However, the Cdc7 or Dbf4-related molecules from higher eukaryotes did not replace the functions of the yeast proteins in simple complementation assays. Therefore, functional assays needed to be conducted to examine whether these molecules actually play similar essential roles in DNA replication of higher eukaryotes. Studies in Xenopus egg extracts showed that an antibody against XeCdc7 inhibited DNA replication in vitro, suggesting an essential role of Xenopus Cdc7 kinase for DNA replication (Roberts et al., 1999). More recently, depletion of Cdc7 from the egg extracts was reported to results in complete loss of replication activity and addition of Cdc7 kinase fractions restores DNA replication (Jares and Blow, 2000; Walter, 2000).

In mammalian cells, injection of an antibody against ASK or huCdc7 protein leads to inhibition of DNA replication in fibroblast cells (Jiang et al., 1999; Kumagai et al., 1999), indicating an essential role of the Cdc7-ASK kinase complex in DNA replication. Essential role of Cdc7 kinase in mammalian cell proliferation was more directly indicated by the fact that mutant mice lacking muCdc7 are early embryonic lethal at E3.5–6.5 (Kim et al., unpublished communications). Apparent survival of early embryos until the blastocyst stage may be accomplished by the maternal stock of muCdc7 protein. Alternatively, some other bypass pathway may be activated in the absence of muCdc7 in early cleaving fertilized eggs. These results establish the conserved essential function of Cdc7-related kinase complexes in DNA replication of higher eukaryotes.

The original characterization of cdc7(ts) in budding yeast indicated that protein synthesis is no longer required for completing S phase once the Cdc7 function is executed (Hartwell, 1974). This suggests that Cdc7 may be the ultimate factor required for initiation of S phase. It is also well established that CDKs are required for cell cycle progression. CDKs complex with cell cycle stage-specific cyclin partners, whose expression levels oscillate during the cell cycle. In G1, G1-specific cyclins (Cln1, 2, 3 in budding yeast and cyclin Ds in mammals) activate the G1 CDK (Sherr, 1994). Different set of cyclins (Clb 5, 6, and cyclin E) are required for initiation of the S phase. It was reported recently that Clb 5, 6-dependent Cdc28 functions are required for execution of Cdc7 functions, showing that G1/S CDK functions prior to Cdc7 in yeast (Nougarede et al., 2000). In contrast, a requirement of Cdc7 function prior to CDK was shown in an in vitro replication Xenopus egg extract system (Jares and Blow, 2000; Walter, 2000). The results indicate that Cdc7 must first function in order for CDK to activate the origins. Both Cdc7 and CDK are required for loading of Cdc45 onto chromatin in yeast, as well as in Xenopus egg extracts (Jares and Blow, 2000; Walter, 2000; Zou and Stillman, 2000).

How can these apparently contradictory results reconcile? Since cell cycle progression in Xenopus egg extracts is an “embryonic-type”, in which S and M phases alternate without G1 phase, requirement for CDK or G1-S transition normally observed in cells with a “somatic-type” cell cycle may be absent. In the latter cells, CDK may need to function twice; first to generate “pre-activated preRC” for Cdc7 action and second to finally activate the replication machinery. MCM2 is phosphorylated by both CDK and Cdc7, and phosphorylation by the former protein is required for recognition of the phosphorylation sites by the latter protein on MCM2 (Masai et al., 2000). Thus, MCM2 may be one of the critical target proteins for both CDK and Cdc7, and sequential action of these kinases may be essential for initiation. To ultimately activate replication, phosphorylation of additional target(s) by CDK may be required. In fact, a number of novel factors associate with Cdc45 and DNA polymerase ε (Araki et al., 1995; Kamimura et al., 1998; Masumoto et al., 2000), both of which are essential for ultimate activation of the replication machinery. It is likely that possible CDK target(s) may be present among these factors.

Cdc7-related catalytic subunits share significant similarity of the primary structure in their kinase domains-about 45% identity between mammals and yeasts (Fig. 2). In contrast, Dbf4-related regulatory subunits are unexpectedly divergent between species (Kumagai et al., 1999; Takeda et al., 1999). No significant overall similarity is detected between budding yeast Dbf4 and fission yeast Dfp1/Him1 except for small stretches of amino acids located close to the C-termini of both proteins. Closer examination of the primary structures of Dbf4-related proteins from various species reveals the presence of two additional conserved stretches (Fig. 2) (Masai and Arai, 2000a). These sequences from the N-terminus were named Dbf4-motif-N, Dbf4-motif-M, and Dbf4-motif-C. Detailed molecular dissection of Dfp1/Him1 and ASK indicates essential roles for motif-M (a novel proline-rich motif) and motif-C (a C2H2-type zinc-finger motif) in mitotic functions and kinase activation (Ogino et al., 2001). In Dfp1/Him1, both a 80 residue segment of motif-M and a 60 residue segment of motif-C individually bind to Hsk1. Kinase activation requires concomitant binding of these two motifs to Hsk1. The segment connecting these two motifs is not conserved and can be varied in length and sequence without compromising in vivo mitotic activity. In vitro, even the two separate polypeptides containing motif-M and motif-C can activate Hsk1 to some extent. Similarly, a small segment of human ASK containing both motif-M and motif-C as well as the presence of two separate polypeptides, each containing motif-M and motif-C, are sufficient for phosphorylation of mammalian MCM2 (Sato et al., unpublished communications). These results indicate a novel mode of kinase activation through bipartite binding of the regulatory subunit to the catalytic subunit (Fig. 3). C-terminally truncated Dbf4 cannot rescue dbf4 null cells, but can complement dbf4(ts), suggesting possible oligomerization of Cdc7-Dbf4 molecules (Dowell et al., 1994; Kihara et al., 2000). Oligomer formation of Cdc7 through self-interaction was reported in budding yeast (Shellman et al., 1998). These results suggest more complex mode of kinase activation, which may involve oligomerization of Cdc7-Dbf4.

Figure 3. Activation of Cdc7 kinase by bipartite binding of Dbf4-related regulatory subunits. Cdc7 kinase is generally inactive on its own, and its kinase activation requires association with the Dbf4 regulatory subunit (represented as three small circles connected by variable linker segments shown as wavy lines). Activation of Cdc7 kinase requires only two small stretches of residues within a Dbf4-related molecule, Dbf4- (motif-M and Dbf4-) motif-C (shown as M and C in circles), both of which can individually bind to Cdc7 catalytic subunit. The segment connecting the two motifs can be deleted or substituted with other sequences without significant effect on the mitotic activity of Dbf4. The Dbf4-motif-N, related to the BRCT motif, is not required for kinase activation or mitotic functions, but is involved in association with replication machinery at the replication origins.

Dbf4-motif-N, related to the BRCT motif (BRCA C-terminal region), is not required for either kinase activation or mitotic functions, but appears to be required for association with chromatin. him1 mutants lacking an motif-N are sensitive to HU and DNA damaging agents (e.g., MMS; Takeda et al., 1999). The fraction of cut cells increases in the presence of HU, whereas the sensitivity to MMS is due to delayed recovery from DNA damage. In one hybrid assay between Dbf4 and replication origin sequences in budding yeast, deletion of motif-N resulted in a loss of specific interaction with the origin (Ogino et al., 2001), suggesting that the motif-N may be involved in the interaction of Dbf4 protein with the replication machinery.

Human Cdc7 kinase can phosphorylate MCM components in vitro (Sato et al., 1997). Genetic interaction in yeasts between Cdc7 and Mcm2, and evidence that MCM2 is phosphorylated in a manner dependent on Cdc7 function in vivo, (Lei et al., 1997; Snaith et al., 2000; Takeda et al., 2001), show the physiological significance of MCM2 as a critical target of Cdc7 kinase. MCM2 is phosphorylated by Cdc7 in mammalian cells, as well as in Xenopus egg extracts (Jares and Blow, 2000; Cho et al., unpublished communications). Thus, MCM2 appears to be one of the most critical target proteins of Cdc7 in all eukaryotic cell types studied.

More genetic evidence for Cdc7-MCM interaction was provided by isolation of the bob1 mutant as a suppressor of cdc7(ts) and its subsequent identification as an allele of MCM5 (Jackson et al., 1993; Hardy et al., 1997). The bob1 mutant protein, in which the conserved proline at position 83 of MCM5 is replaced by leucine, is a bypass suppressor for Cdc7 and Dbf4 functions, since it can suppress null mutants of both cdc7 and dbf4. Although the mechanism of bob1-mediated bypass of Cdc7 functions is not clear, it strongly indicates that crucial roles of Cdc7 kinase are linked to the MCM complex.

MCM was originally identified as being required for stable maintenance of ARS plasmids in budding yeast (Maine et al., 1984; Sinha et al., 1986; Tye, 1999). Later, “licensing factor”, which is required for one-time replication of the eukaryotic chromosome, was purified from Xenopus egg extracts and identified as the Xenopus homologue of MCM (Kubota et al., 1995; Thommes et al., 1997). MCM is essential for initiation of DNA replication and is associated with chromatin before S phase initiation, but it is quickly inactivated or sequestered from chromatin after initiation, thus being precluded from reutilization for origin activation within the same S phase (Tye, 1999). MCM is loaded onto chromatin at late M to G1 phase in yeasts. It dissociates from chromatin after DNA replication is initiated in both yeast and Xenopus egg extracts. Precise biochemical functions of MCM were not previously known. Ishimi (1997) purified MCM from HeLa cells and discovered DNA helicase activity in the dimeric MCM4-6-7 complex. The processivity of this helicase activity is limited in displacing non-tailed DNA, but has high processivity on tailed DNA by binding to a forked DNA structure as a double hexamer (Lee and Hurwitz, 2001; You et al., unpublished communications). Interestingly, no helicase activity was detected in the MCM2-4-6-7 complex or the MCM2-3-4-5-6-7 complex (Adachi et al., 1997; Ishimi et al., 1998). The active helicase is composed of a dimer of trimeric MCM4-6-7 and forms a ring-like structure, similar to that observed in many hexameric helicases. MCM2 inhibits helicase activity of MCM4-6-7 by forming a tetrameric complex (Ishimi et al., 1998; Sato et al., 2000). Since MCM components migrate along the chromosome as the replication forks migrate away from the origins (Aparicio et al., 1997), MCM is likely to be a part of the replicative DNA helicase at eukaryotic replication forks.

MCM2 is efficiently phosphorylated in vitro by Cdc7 kinase from budding yeast, fission yeast, and human (Lei et al., 1997; Sato et al., 1997; Brown and Kelly, 1998; Jiang et al., 1999; Kumagai et al., 1999; Takeda et al., 1999; Kihara et al., 2000; Masai et al., 2000; Takeda et al., 2001; Ishimi et al., 2001). In human, both the free form of MCM2 and the MCM2-4-6-7 complex form are efficiently phosphorylated in vitro by huCdc7. MCM4 and MCM6 in the MCM2-4-6-7 complex are also phosphorylated in vitro by huCdc7-ASK to a lesser extent. Differential mobility-shifts on SDS–PAGE after phosphorylation by Cdc7 indicate phosphat ion of selective residues on MCM2 in the complex (Masai et al., 2000). Tryptic peptide mapping also confirmed phosphorylation of distinct residues on MCM2 in the complex (Cho et al., unpublished communications). Kihara et al. reported that prior dephosphorylation of the MCM2 substrate leads to loss of phosphorylation by Cdc7 (Kihara et al., 2000), suggesting that prior phosphorylation of the MCM substrate is required for target site recognition by Cdc7. This is also the case for mammalian MCM2 protein (Masai et al., 2000). The efficacy of MCM2 phosphorylation by Cdc7 is significantly increased when the substrate is pre-phosphorylated by CDK2. This stimulation occurs with MCM2 in the MCM2-4-6-7 complex, but not with uncomplexed MCM2. These results indicate that CDK and Cdc7 collaborate to achieve efficient phosphorylation of critical residues on MCM2 in the complex for initiation of DNA replication.

Although the precise architecture of eukaryotic replication forks is not known, MCM is likely to play a major role in advancing their action. The hexameric MCM2-3-4-5-6-7 complex has no detectable helicase activity (Adachi et al., 1997) and must be converted to an active helicase prior to initiation of DNA chain elongation (Fig. 4). This can be achieved by reorganization of the subunit assembly or by association with other proteins. However, the purified mammalian MCM2-4-6-7 complex cannot be converted to an active helicase simply on phosphorylation by huCdc7 in vitro (Masai et al., unpublished communications). Origin activation by Cdc7 may require MCM3-5 subunits and/or other pre-replicative components such as Cdc45. The loading of Cdc45 onto chromatin requires both Cdc7 and CDK (Jares and Blow, 2000; Walter, 2000; Zou and Stillman, 2000). The phosphorylation of MCM by Cdc7 and/or CDK may trigger its association with Cdc45 directly or through other proteins, and this may bring DNA polymerases onto the preRC (Uchiyama et al., 2001a,b).

Figure 4. A model for activation of MCM complexes by phosphorylation. Accumulating evidence points to the essential role of MCM in duplex unwinding at the replication forks. Cdc7 is not required for loading of MCM onto preRC, but is essential for activation, which presumably induces local conformation change of the origin DNA. Since MCM2-4-6-7 and MCM2-3-4-5-6-7 is inactive as a helicase, reorganization of the MCM assembly may take place upon phosphorylation by the Cdc7 kinase. The upper pathway proposes a possibility that upon phosphorylation events MCM2 and 3-5 complex, which is inhibitory for MCM4-6-7 helicase activity, may dissociate from the MCM2-3-4-5-6-7 complex within the preRC, leading to activation of the MCM4-6-7 helicase. Alternatively, phosphorylation by CDK and Cdc7 may induce critical conformational change of the MCM2-3-4-5-6-7 complex and/or association of Cdc45 or other helicase accessory proteins with preRC, leading to generation of an active and processive helicase at the fork (lower pathway).

In budding yeast, functioning of Cdc7 kinase is associated with generation of specific nuclease hypersensitive sites near the origin sequences (Geraghty et al., 2000). This suggests that phosphorylation of a specific target protein(s) leads to the structural change of the origin required for initiation of DNA replication. Such Cdc7-dependent structure changes are prematurely observed in G1 phase in the bob1 mutant, suggesting that Cdc7-MCM interactions regulate this structural transition at the onset of DNA replication. This conformational change of DNA most likely represents partial unwinding of the origin DNA, since RPA, the eukaryotic SSB protein, is associated with DNA after Cdc7 functions are executed (Tanaka and Nasmyth, 1998).

Generally, MCM dissociates from chromatin after S phase is initiated (Fujita et al., 1996, 1998), and this may be associated with phosphorylation of MCM subunits. Extensive phosphorylation of MCM4 by Cdc2 kinase during G2-M phase may prevent re-association of MCM with chromatin until the next G1 phase (Fujita et al., 1998). Another level of regulation may be direct inhibition of the MCM4-6-7 helicase by phosphorylation of MCM4 by CDK2-Cyclin A (Hendrickson et al., 1996; Ishimi et al., 2000; Ishimi and Komamaura-Kohno, 2001). The precise roles of these phosphorylation events in regulation of origin firing need to be investigated by the use of functional in vitro assay systems measuring activation of DNA replication from specific origin sequences.

ROLE OF CDC7 IN DNA REPLICATION CHECKPOINT CONTROL AND MAINTENANCE OF CHROMATIN STRUCTURES DURING S PHASE

Figure 5. Possible roles of the Cdc7 kinase in various aspects of cell cycle control of eukaryotic cells. Yeast genetic studies reveal various interactions (double-headed arrows), which suggest possible roles for the Cdc7 kinase in regulation of cell functions other than mitotic DNA replication. The figure is based on the available genetic data mainly obtained from yeast studies and is not meant to indicate that Cdc7 has conserved roles in all the eukaryotes in the cell cycle events indicated in this figure.

Budding yeast Dbf4 and fission yeast Dfp1/Him1 are hyper-phosphorylated in response to S phase arrest by HU, and this hyper-phosphorylation depends on Rad53 and Cds1, respectively (Brown and Kelly, 1999; Takeda et al., 1999; Weinreich and Stillman, 1999). Similar hyper-phosphorylation is detected on Hsk1 protein under the same conditions (Snaith et al., 2000). Cds1 can phosphorylate Hsk1 and Dfp1/Him1 in vitro. Cds1-dependent hyper-phosphorylation of Dfp1/Him1 protein is not observed in hsk1-89, consistent with the requirement of Hsk1 for checkpoint responses. These results indicate that Hsk1 may be involved in signal transduction of the DNA replication checkpoint. Kihara et al. reported that Cdc7-Dbf4 kinase is inactivated by phosphorylation of Dbf4 subunit by Rad53 kinase in vitro (Kihara et al., 2000). This mechanism may inhibit further origin activation after HU-mediated S phase arrest. Down-regulation of cellular Cdc7 kinase activity after HU arrest also occurs (Weinreich and Stillman, 1999). The precise roles of Hsk1 and Cds1-dependent phosphorylation of Dfp1/Him1 in DNA replication checkpoint await analyses of mutant Hsk1 and Dfp1/Him1 proteins specifically defective in checkpoint responses.

Under some conditions, hsk1(ts) mutants display abnormal nuclear morphology with an apparent defect in mitosis (Takeda et al., 2001). Double mutants of hsk1-89 and rad21 (encoding a subunit of the cohesion complex) exhibit significant growth retardation even at permissive temperatures and show similar abnormal nuclear structures (Fig. 5). Premature separation of the replicated daughter chromosomes occurs in a significant population of hsk1-89 cells, suggesting that sister chromatid cohesion is partially lost in the hsk1 mutant. Similar abnormal chromosome morphology is also seen in another allele of hsk1(ts) (Snaith et al., 2000). Reduced origin firing in hsk1-89 may indirectly affect the rate of sister chromatid cohesion, since cohesion may be tightly coupled to DNA replication. Alternatively, Hsk1 kinase may more directly regulate the processes of sister chromatid cohesion.

Characterization of cdc7(ts) in budding yeast revealed its role in meiosis (Sclafani et al., 1988). Sporulation is arrested in cdc7(ts) diploid cells, presumably due to impaired synaptonamal complex formation. In fission yeast, hsk1+ is also required for meiosis (Ogino et al., submitted). Nitrogen starvation of a hsk1(ts)/hsk1(ts) diploid strain results in formation of aberrant spores containing only one asci. Analysis of DNA content after nitrogen starvation indicates that premeiotic DNA replication is completely blocked in the hsk1(ts)/hsk1 diploid even at 25°C, a permissive temperature for this mutant, indicating that Hsk1 also plays a crucial role in initiation and/or progression of premeiotic DNA replication. Similar mei phenotypes were observed also in a mutant of him1/dfp1 (Ogino et al. unpublished communications), suggesting that Hsk1-Him1/Dfp1 kinase is required for the precesses of meiosis. In mammals, both Cdc7 and ASK mRNAs are abundantly expressed in testis, consistent with conserved essential functions of Cdc7 kinase during meiosis (Sato et al., 1997; Jiang et al., 1999; Kumagai et al., 1999).

Both muCdc7 mRNA and protein are detected in adult brain (Jiang and Hunter, 1997; Sato et al., 1997; Kim et al., 1998). Brain muCdc7 polypeptides include some distinct from those detected in other tissues. The ASK regulatory subunit is not detected in brain, either at transcript or protein levels (Kim et al., unpublished communications). What role, if any, does Cdc7 kinase play in non-proliferating brain cells, and is there a brain-specific activator for Cdc7 kinase? Generation of brain-specific knockout of Cdc7 and biochemical characterization of brain Cdc7 proteins will be required to answer these questions.

Cdc7-related kinases are characterized by the presence of so-called “kinase insert” sequences that interrupt the conserved kinase domains at specific structural boundaries. The insert sequences of Cdc7 kinase family are found between kinase domains I and II, VII and VIII, and X and XI (Hanks et al., 1988), although their lengths and sequences are generally less conserved compared to kinase domains. In fission yeast, there is another kinase structurally related to Cdc7 (Table 1). spo4+ encodes a serine-threonine kinase, which shares 60% sequence identity with budding yeast Cdc7 and contains two kinase insert sequences at the conserved positions (Nakamura et al., 2002). Unlike hsk1+, spo4+ is not essential for vegetative growth, but the mutant strain shows a defect in sporulation. In the same set of the mutant strains, spo6+ was identified as a Dbf4-related molecule (32% identical to Dfp1/Him1) containing the three conserved motifs described above (Masai and Arai, 2000a; Nakamura et al., 2000). Further studies revealed that Spo6 and Spo4 form an active kinase complex (Nakamura et al., 2002). These results indicate the presence of members of the Cdc7-Dbf4 protein family which play distinct roles in cell proliferation and differentiation. It remains to be seen whether mammalian cells contain additional Cdc7-Dbf4 family members.

It is now firmly established that Cdc7-Dbf4 is an essential cell cycle-regulated kinase complex that is structurally and functionally conserved in eukaryotes (Masai et al., 2000). Cdc7 is activated by association with the regulatory subunit Dbf4, similar to cyclin-dependent activation of CDK, although the structural basis for this activation may be distinct from that of CDK. Expression of the regulatory subunit, and thus its kinase activity, is cell cycle-regulated. This serves as a molecular switch for DNA replication by phosphorylating specific subunits of the MCM complex on the chromatin. Upon phosphorylation by Cdc7, MCM is likely to be converted to an active form at the replication origin, which together with CDK induces the unwinding of the DNA and the loading of other replication proteins including Cdc45 and DNA polymerases (Fig. 4). Crucial issues yet to be resolved are the molecular mechanisms by which the Cdc7 kinase induces the transition from preRC to the fired complexes. Development of an enzymatic assay system to measure Cdc7-induced origin firing on a defined template DNA containing specific origin sequences is key to solving this problem.

Cdc7-Dbf4 may also play an important role during checkpoint responses induced by arrested replication forks. A sufficient level of Cdc7 kinase activity is required for Rad53/Cds2 activation in response to replication fork arrest by nucleotide deprivation. A possible role of Cdc7-Dbf4 as a target of intra-S checkpoint regulation was suggested from analyses using the Xenopus egg extract DNA replication system (Costanzo et al., 2000; Jares et al., 2000).

Additional functions of the Cdc7-Dbf4 kinase in the meiotic cell cycle are known from genetic studies in yeast, although it is not clear whether the target(s) of Cdc7 in meiosis are distinct from those in the mitotic cell cycle. Even more ambiguous is its role in UV-induced mutagenesis (Hollingsworth et al., 1992) and in maintenance of sister chromatid cohesion during S phase. Future biochemical and genetic studies will shed light on possible novel functions of the Cdc7 kinase during cell cycle regulation.

Finally, the likely multiple Cdc7-Dbf4-related kinase complexes in eukaryotes may point to the possibility that Cdc7-Dbf4 forms a novel kinase family, each member of which plays a distinct role in regulation of mitotic and meiotic cell cycle progression.

We thank Dr. Gerard Zurawski (DNAX Research Institute) for critical reading of and valuable suggestions on the manuscript. We thank the members of our laboratory for their contribution to the works described in this review.